Dihydropteroylglutamic acid and process for preparing the same



Patented Mar. 13, 1951 UNITED STATES PATENT OFFICE'DIHYDROPTEROYLGLUTAMIC ACID. AND PROGESS FOR PREPARING- THE SAME Boyd.L. ODell, Columbia, Mo.-,-assignr to Parke, 1Davis & i Company, Detroit,Mich., a corpora- '.tion (of Michigan No Drawing. :lAp'plicationSeptember-14; 1946, .e Serial No. 697,155

' 3TClaims. f (Cl. 260-:2515) .The invention relateseto. the preparation.of dium). ifI'he mixtureis-shaken ina hydr ogenaneworganic compoundswhich areusefulin nutionaapparatus like-.that described by Smith, J.trition and therapy and also as intermediates for Biol. Chem, 96'; 35..(-19.32); in. an atmosphere. of the preparation of vitamin Be and itsderivatives. hydrogen for about 6 hours and the total amountMoreparticularly, the invention relates (to the of hydrogen absorbeddetermined-'at the end'of preparation of dihydro-vitamin Be, otherwisethis time. .In this-example. almostjexactlythe known as.dihydropteroylglutamic acid. calculated amount of hydrogen (;1 mole) istaken Vitamin Be isan antianemia factor and can. be up toconvert thevitamin (1"m'ole) with-empiriobtained from various naturalstartingmaterials, cal formula, CisiHmOsNmunto' the new 'dihydrosuch as livertissue or yeast, by hydrolytic, enzy- 10 vitamin Be(dihydropteroylglutamic acid) of matic or like treatment of suchmaterials, folformula, C19H2106N7. Duringthe hydrogenation lowed byisolation,procedures. Vitamin .Be .is the. yellow; colorrofthe-originalfsolution,gradu- N- [4-'( (2-amino-4-hydroxy 6 -.pteridyl)methally fades to :giveaclear-colorless solution. '..Thns.

yllamino)b enz oyll r glutamic acid, otherwise the new; dihydro.compound .is.5colorless,:.asdisknown as pteroylglutamic. acid, and maybe.reptinguishedfromthe .vitaminitself: whichiisieolresentedt by theformula, ,ored yellow. However, the dihydrdcom-pound H N-C CN=..O-.CH2NHv 5 a,

NN-N=CH v Vitamin Be I have. now found that dihydro.-'N.-[4'-([( 2quickly turns brown -uponiexposureto air or aminoi-hydroxy 6pteridyl)methyl-lamino)- oxygen. benzoyll glutamic. acid can be preparedby chem- While excluding air as 'much asj'possible; the

ical methods and is an.exdellentlintermediatealkalineliydrogenation"solutioniis filterd'from for. -the. preparationof. vitamin Be .itselfas =well the-catalyst and the filtrate immediatelyadjusted as for the preparation of conjugates of the latter to pH. 3.0by dilute hydrochloric acid. This with. more. glutamic acid residues andother decauses the"dihydro vitaminlBeito,precipitatein rivatives ofvitamin Be. I have also found that, fine micro-crystalline form. Theprecipitated difor practical purposes the new .dihydro-product.hydro-compoundis washedfour timeswith' 0.01 can be administered directlyto the animal or N'hydroch'loric acid'andonce'With water. "It'liS humanorganism-with results comparable in n-uthen dried over calcium chloridein a vacuum merous instances to those obtained when admindesiccator."F01" analysis -itJis""fi i rther. dried" at istering vitamin Beitself.This-is probably due 145;Ciin ahigh vacuum toconstant weightxand to the,fact that the dihydro compound readily gives thefollowing.analyticalifigures.

oxidizesin air to vitamin Be. "In' preparing my new dihydrovitamin"Be" Ii g iP 2$ can start withrelatively...simplechemicals and 49 u d, 0synthesizeethe dihydro compound. ..Alternatively, C 51397 4 I can startwith the isolated vitamin Be and di- 51'16 rectly hydrogenate it to getdihydro-vitamin Bo.

The invention will be understood from the The u trav l absorption urv ofv tam n following examples. Be in alkaline solution has maxima at 255,282

and 365 m i but the curve of the new dihydro- Example 1 vitamin Be ofthis example in alkaline solution Y Y has only one maximum, located at284 m with 1 9g-m l ra s f pu dry vitam B pt molecular extinction of21,300 at pH 11.0. The glut'amic acid). pr p d for ple as defollowingExample 2 shows thejrelationship of scribed by Pfifiner et al. inScience, volume 97, the new dihydro-compound to vitamin Be. page 404(1943), are dissolved in 15 cc. of 0.1 N

sodium hydroxide solution and put in a micro-, Example 2 hydrogenationapparatus with 200 milligrams of 10' milligrams of pure drydihydro-vitamin Be palladium-barium sulfate catalyst (5% palla- 55(dihydropteroylglutamic acid) from Example 1 are dissolved in 15 cc. of0.1 N sodium hydroxide solution and shaken at room temperature with adefinite volume of oxygen in a closed system for 3 hours. The oxygenuptake is then measured and is found to be almost exactly the amountrequired to" oxidize (dehydrogenate) 10 milligrams of dihydro-vitamin Beof formula CmHzrOeNw into vitamin Bo of formula The yellow alkalineoxidation solution is brought to pH 3.0 by adding thereto 1.0 Nhydrochloric acid. This precipitates the vitamin Be which is collected,washed three times with water, and dried. The dry product is dissolvedin 1 N hydrochloric acid and any small amount of insoluble materialremoved from the solution which is thereafter adjusted to pH 3.0 withdilute alkali. The vitamin again precipitates, is filtered 01f,recrystallized from hot water and dried over calcium chloride.Foranalysis, it is dried at 145 C. in a high vacuum. The product is ayellow crystalline compound identical with the starting material used inExample 1 and having the ultraviolet absorption characteristics ofvitamin B0. Analytical figures for the dry product are:

Calculated. for C19H1906N7 Z C, 51.71%; H, 4.34% Found:

By running this oxidation example in the presence of the hydrogenationcatalyst used in Example 1, only 1 hour, instead of 3 hours, is neededto completely convert thedihydro-vitamin B to the vitamin.

Example 3 14.1 grams of 2,4,5-triamino-6-hydroxypyrimidine (C4H7ON5) aredissolved, along with 26.6

grams of p-aminobenzoyl-1(+)-glutamic acid (C12H14O5N2), in 2 liters ofhot distilled water which has been boiled to remove air and in which isdissolved a buffer mixture consisting of 12.3 grams of sodium acetate(NaOOCCI-Ia) and .9 grams of acetic acid (HOOCCHs) The hot solution ofthe triamine and the amino acid is filtered to remove any small amountof solids or undissolved materials which may be present, the filtratecooled, and then 21.6 grams of 2,3-dibromopropionaldehyde addedgradually While stirring vigorously. All of the foregoing operations arecarried out in a closed system with strict exclusion of oxygen or air,e. g. in an atmosphere of nitrogen.

After the reaction mixture is stirred for 24 hours, it is brought to pH3.0 by the addition of air-free 1.0 N hydrochloric acid. Thisprecipitates dihydro-vitamin Bo (dihydropteroylglutamic acid) which isfiltered off, washed three times with air-free 0.01 N hydrochloric acidand once with air-free water and then dried. All of these operations arein the absence of air or oxygen.

The dihydro-vitamin of thisexample, like that of Example 1, can readilybe oxidized to the vitamin as described under Example 2 for the dihydrocompound of Example 1.

Example 4 2 grams of sodium acetate and 2.66 grams of p aminobenzoyl1(+) -glutamic acid are dissolved in 300 cc. of air-free distilled waterwhile blanketing the solution with nitrogen. Nitrogen is bubbled throughthe solution and 1.4 grams of a-bromoacrolein are added with stirring.The mixture is stirred for15 minutes and the resulting solution added toa solution of 1.55 grams of 2,4,5-triamino-6-hydroxypyrimidine in 200cc. of air-free water. A precipitate separates from the "reactionmixture a short time after the reactants are mixed together. Thereaction mixture is shaken under nitrogen for 48 hours and is thenbrought to a pH of 3.0 by addition of air-free 1.0 N hydrochloric acid.The dihydro-vitamin Be (dihydropteroylglutamic acid) which separatesupon adding the hydrochloric acid is filtered off, washed twice withair-free 0.01 N hydrochloric acid and once with air-free distilled waterand then dried. All of the foregoing operations are conducted withstrict exclusion of air or oxygen. The dihydro-vitamin Be product ofthis example, like those of Examples 1 and 3, has the empirical formulaC19H2106N'7 and can be readily oxidized to vitamin B6.

7 In Example 1 above, the catalytic hydrogenation of vitamin Be to itsdihydro derivative under alkaline conditions is described whereinpalladium metal is used as a catalyst. Instead of using palladium, anyother noble metal catalyst, or combinations thereof, may be used whichare capable of catalyzing the addition of two atoms 3. The compound,dihydropteroylglutamio acid.

BOYD L. ODELL.

REFERENCES CITED The following references are of record in the file ofthis patent:

UNITED STATES PATENTS Name Date Waller et al Mar. 14, 1950 OTHERREFERENCES Liebigs Annalen, vol. 548, pp. 284-289.

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3. THE COMPOUND, DIHYDROPTEROYLGLUTAMIC ACID.